This is the competitive renewal application for the AI-41236 grant. In the past 5 years, we have studied suppression of autoimmune ovarian disease (AOD) and autoimmune prostatitis (EAR) by the polyclonal CD4+CD25+ regulatory T cells (Treg). Disease-specific Treg were found to accumulate in regional LN where they reversibly suppressed host T cell response. We have now observed that even in normal mice, disease-specific Treg are highly enriched in the regional LN;thus, the ovarian LN Treg preferentially suppress AOD. In Aim 1, we will test the hypothesis that Ag-specific Treg respond to tissue Ag in the regional LN, gain disease-specific Treg function, and collectively maintain physiological tolerance. We next discovered that rapid functional acquisition and disease suppression by polyclonal Treg were both dependent on endogenous Ag exposure, and that only Ag-experienced Treg retained disease-suppressing function in an Ag-negative host. Thus, in Aim 1, we will investigate the existence of naive and memory polyclonal Treg, their life span, and the thymic influence on the maintenance of naive Treg function. For the second major project, we have developed a model of transgenic autoimmune orchitis (TAO) in mice that co- expressed transgenic ovalbumin (OVA) in the male haploid germ cells and pOVA specific transgenic T cell receptor (DO11.10). TAO that developed spontaneously remained for 10 weeks in a preclinical state with OVA autoAb, focal non-invasive orchitis and normal fertilty. At ~17 wk, TAO transitioned into severe invasive orchitis that eliminated testicular function and rendered the animals infertile. The T cells from each stage of TAO, transferred orchitis to OVA+ rag knockout recipients with severity that matched the TAO of the cell donor. In proposed Aim 2, we will investigate the following mechanisms of TAO progression: 1) Discharge of OVA and testis-derived Toll-like receptor ligands (HSP and protamine /nucleotide complex) via the phagosomes of Sertoli cells (the residual bodies) to the intersitital space, 2) Stimulation of antigen presenting cells and OVA-specific T cells outside the blood-testis barrier, and 3) Silencing of OVA-specific Treg. By exploiting appropriate autoimmune disease models, we aim to elucidate important cellular mechanisms whereby the autoimmune process is normally controlled, and how clinical autoimmunity is induced.